Emergence of KPC-2 and NDM-5-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae with high-risk sequence types ST11 and ST15

ABSTRACT The emergence of Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-β-lactamase (NDM)-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae (KPC-2-NDM-hv-CRKP) poses a certain threat to public health. Currently, only a few sporadic reports of such double-positive hv-CRKPs were available. In this study, we isolated two KPC-2-NDM-5-hv-CRKPs from elderly patients with serious underlying diseases and poor prognoses. We found both FK3122 and FK3127 were typical multidrug-resistant (MDR) isolates, exhibiting high-level resistance to both carbapenems and novel β-lactamase inhibitors ceftazidime/avibactam. Notably, FK3122 is even resistant to cefiderocol due to multiple blaNDM-5 elements. Besides the MDR phenotype, A549 human lung epithelial cells and Galleria mellonella infection model all indicated that FK3122 and FK3127 were highly pathogenic. According to the whole-genome sequencing analysis, we observed over 10 resistant elements, and the uncommon co-existence of blaKPC-2, blaNDM-5, and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: rmpA and iuc operon. We also found that the resistance and virulence plasmids in FK3127 could not only be transferred to Escherichia coli EC600 independently but also together as a co-transfer, which was additionally confirmed by the S1-pulsed-field gel electrophoresis plasmid profile. Moreover, polymorphic mobile genetic elements were found surrounding resistance genes, which may stimulate the mobilization of resistance genes and result in the duplication of these elements. Considering the combination of high pathogenicity, limited therapy options, and easy transmission of KPC-2-NDM-5-hv-CRKP, our study emphasizes the need for underscores the imperative for ongoing surveillance of these pathogens. IMPORTANCE Hypervirulent Klebsiella pneumoniae drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP). However, little information is available on the virulence characteristics of the New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase-2 (KPC-2) co-producing K. pneumoniae strains. In this study, we obtained two KPC-2-NDM-hv-CRKPs from elderly patients, each with distinct capsule types and sequence types: ST11-KL64 and ST15-KL24; these ST-type lineages are recognized as classical multidrug-resistant (MDR) K. pneumoniae. We found these KPC-2-NDM-hv-CRKPs were not only typical MDR isolates, including resistance to ceftazidime/avibactam and cefiderocol, but also displayed exceptionally high levels of pathogenicity. In addition, these high-risk factors can also be transferred to other isolates. Consequently, our study underscores the need for ongoing surveillance of these isolates due to their heightened pathogenicity, limited therapeutic options, and potential for easy transmission.

KEYWORDS Klebsiella pneumoniae, NDM-5, KPC-2, hypervirulent K lebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), which are encoded by the bla KPC and bla NDM genes respectively, were two major carbapenemases in Enterobacteriaceae, especially in Klebsiella pneumoniae (1).These carbapenemases are capable of hydrolyzing not only carbapenems but also most other important β-lactam antibiotics, making them a significant challenge to clinical treatment (2).Carbapenem-resistant K. pneumoniae (CRKP) was listed as the most urgent antibiotic resistance threat (1).On the other hand, a different type of Klebsiella pneu moniae known as hypervirulent K. pneumoniae (hvKP) mainly infects healthy individu als and can cause severe disseminated infections (3).Recently, an increasing number of K. pneumoniae strains have been identified that combine hypervirulent and multi drug-resistant characteristics, referred to as hv-CRKP (4).However, the hypervirulent K. pneumoniae strains that co-produce NDM and KPC-2 carbapenemases were rare to find.
In this study, we not only featured the antibiotic-resistant and hypervirulent phenotypes in detail, but we also investigated the molecular mechanisms associ ated with multidrug resistance and hypervirulent phenotypes using whole-genome sequencing (WGS).We also analyzed the plasmid-backbone and conjugation modules and used a conjugation assay to further determine the self-transmissibility of these resistance and hypervirulence genes to study how these isolates emerged and the risk of dissemination of these isolates.According to our findings, the KPC-2-NDM-hv-CRKP poses a significant threat to healthcare networks, and steps should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

Bacterial strains
Both K. pneumoniae strains FK3122 and FK3127 were isolated from elderly patients with serious underlying diseases and poor prognosis.The patient infected with the FK3122 was diagnosed with severe encephalic infection and the patient infected with the FK3127 was diagnosed with severe pulmonary infection.Both strains exhibited typical hypervirulent and carbapenem-resistant phenotypes.K. pneumoniae strain 1627 (classical K. pneumoniae, cKp, ST11) and FK3036 (classical K. pneumoniae, cKp, ST15) were used as virulence-negative control strains, and NUTH-K2044 (ST23-KL1) (10) was used as a virulence-positive control strain.

Antimicrobial susceptibility test
According to the Clinical and Laboratory Standards Institute guidelines, the minimal inhibitory concentration (MIC) of the K. pneumoniae strains FK3122 and FK3127 was determined by broth microdilution.We used Escherichia coli ATCC 25922 as a quality control strain for determining MICs.The interpretative breakpoints were based on CLSI2022-M100-ED31.The cefiderocol breakpoints were based on EUCAST 2022.

Quantitative siderophore production assay
To assess the ability of bacterial supernatants to chelate iron, the researchers utilized the chrome azurol S (CAS) assay, following established protocols (15).Specifically, 10 mL of stationary-phase, iron-chelated cultures was deposited on CAS plates, and after 48 hours of incubation at 37°C, the presence of orange halos was used to identify siderophore production.

Cell culture and K. pneumoniae infection
A549 lung cells (ATCC CCL-185) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a 5% CO 2 incubator.Cells were seeded in 24-well plates at a density of 1 × 10 5 cells per well and incubated for 24 hours.The cell-culture medium was then supplemented with 2 × 10 7 CFU of K. pneumoniae and incubated for 21 hours.After centrifugation at 3,000 rpm and 4°C for 5 minutes, the supernatant was analyzed for lactate dehydrogenase (LDH) using the LDH Cytotoxicity Assay kit (Solarbio BC0685), following the manufacturer's instructions.

G. mellonella in vivo infection model
To assess the pathogenicity of K. pneumoniae strains FK3122 and FK3127, we conducted G. mellonella infection assays.Caterpillars weighing between 150 and 200 mg were selected after storage at 4°C.Treatment groups were inoculated with 10 µL of the bacterial suspension containing 1 × 10 6 CFU/mL, while the control group received normal saline.Each treatment group had at least 30 caterpillars, divided into three Petri dishes, and maintained at 37°C.Survival rates were recorded for 4 days with daily observations.

Conjugation assay
We used a conjugation assay to test if the resistant plasmids from K. pneumoniae FK3122 and FK3127 could be transferred or co-transferred to E. coli EC600 (recipient isolate).Donors and recipients were cultured to logarithmic phase, mixed in a 1:1 ratio, centrifuged at 8,000g for 1 minute, and resuspended in 20 µL of 10 mM MgSO 4 .The resuspension was plated on LB agar and incubated at 37°C overnight.The number of transconjugants per donor was calculated to determine the conjugation frequency.PCR was performed to confirm the presence of the bla KPC-2 , bla NDM-5 , and iuc genes in all transconjugants, using primers listed in Table S1.

S1-pulsed-field gel electrophoresis assay
To confirm plasmid presence in K. pneumoniae FK3127 and its transconjugants, S1pulsed-field gel electrophoresis (S1-PFGE) was used.All PFGE plugs were prepared and digested using a previously described method, then separated for 19 hours on a 1.0% agarose gel using XbaI-digested Salmonella H9812 DNA as a marker.The CHEF mapper system (Bio-Rad) was used for PFGE analysis, and DNA in the gels was stained with Gel-red (Yeasen, China) at 6 V/cm and 14°C with pulse times ranging from 4 to 40 seconds.S1 nuclease endonuclease (Takara, Dalian, China) was used to treat the isolates, which were embedded in 10 g/L Seakem Gold gel before PFGE analysis.

Nucleotide sequence accession numbers of K. pneumoniae FK3122 and FK3127
The complete nucleotide sequences of the K. pneumoniae FK3122 and FK3127 were submitted to GenBank under accession numbers JAOVSH000000000 and JAOVSI000000000, respectively.

Statistics
Statistical significance was assessed using a two-tailed Student's t-test and log-rank (Mantel-Cox) test of the GraphPad Prism9 software.P < 0.05 was considered statistically significant.

K. pneumoniae FK3122 and FK3127 exhibited multidrug resistance pheno type
According to the antibiotic susceptibility test, we discovered that K. pneumoniae FK3122 and FK3127 exhibited similar multidrug-resistant profiles (Table 1); they revealed extensive resistance to all β-lactam antibiotics, including carbapenems, as well as the new β-lactamase inhibitors ceftazidime/avibactam (CZA).Notably, in addition to being resistant to ceftazidime/avibactam, the FK3122 was even resistant to cefiderocol, both of which were recognized as the new effective agents to defend the multidrug-resistant isolates (Table 1).Moreover, the antibiotics that can act against the infection caused by FK3127 were also limited, since it also exhibited high-level resistance toward aminogly coside antibiotics.These antibiotic-resistant phenotypes were threatened for clinical treatments.

K. pneumoniae FK3122 and FK3127 co-harboring bla KPC-2 , bla NDM-5 , and hypervirulent plasmids
We found that FK3122 belonged to ST11-KL64 isolates and FK3127 belonged to ST15-KL24 isolates, both ST-type lineages in which they belong were associated with the CRKP epidemic.In these isolates, we discovered more than 10 resistant elements   as well as several key resistance plasmids (Table 2).The co-existence of bla KPC-2 and bla NDM-5 was studied because it played a pivotal role in conferring resistance to carbape nems and ceftazidime/avibactam.Notably, in the FK3122, we observed four copies of bla NDM-5 , located in the chromosome, and two plasmids, respectively, which may account for its resistance to cefiderocol.Moreover, the presence of rmtB in FK3127 conferred higher-level resistance to aminoglycoside antibiotics (Table 2).Importantly, most of the resistance genes were carried by plasmids and were flanked by numerous mobile elements, heightening the risk of transmission.The bla KPC gene was carried by the classical Tn1721 transposon ( 16), and the rmtB was surrounded by the IS26 islands ("IS26-Tn3-IS6100-mph(A)-IS26-Tn3-bla TEM-1B -rmtB"), which was different from previous findings (17).
Although the plasmids identified in the FK3122 seemed less transmissible, the movement of insertion sequence IS26 surrounding the bla NDM-5 genes was active.IS26 is important in the spread of antibiotic resistance genes in Gram-negative bacteria, forming regions with multiple antibiotic resistance genes flanked by and interspersed with copies of IS26 (21).In FK3122, we observed four copies of bla NDM-5 genes.The conservative bla NDM-5 resistant islands were "IS26-bla NDM-5 -ble MBL -trpF-dsbD-ISCR1-sul1-qacE-addA2-dfrA12-IS26" (Fig. 2C).The duplication of bla NDM-5 was produced by an IS26-formed composite transposon, which was presumably activated by ISCR1, which uses a rollingcircle mechanism and can promote tandem duplication by homologous recombination (21).Moreover, the Tn3-derived inverted-repeat transposable elements were also been found to mobilize bla NDM-5 .These results suggested that mobile genetic elements played an important role in the mobilization of bla NDM-5 .

DISCUSSION
The emergency of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses devastating challenges to public health.Of the epidemiology, most hv-CRKPs were associated with the bla KPC-2 genes and ST11-KL64 isolates (22).To date, only a few occasional reports of strains co-producing NDM and KPC-2 are available.When K. pneumoniae strains combine hypervirulence and carbapenem resistance, clinical treatment becomes complex, and available antibiotics are limited.When hv-CRKP co-harbors NDM and KPC-2, the treatment outlook becomes even worse, as CZA is rendered ineffective due to NDM carbapenemase production.In this study, we characterized two hv-CRKPs that co-harbored NDM, KPC-2, and virulence plasmid.Notably, these isolates derive from two pandemic CRKP lineages, ST11 and ST15.
Epidemiological data indicated that ST11-KL64-hvCRKP has emerged as the most prevalent hypervirulent and carbapenem-resistant K. pneumoniae, and usually contrib uted to the hospital outbreaks of infection (4,22).FK3122 was a typical ST11-KL64-hvCRKP with the presence of IncHI1B virulence plasmid p3122-5 and IncFII bla KPC-2 plasmid p3122-6.However, unlike the classical IncFII bla KPC-2 plasmid and IncHI1B virulence plasmid, both two plasmids were identified to lose the mobile ability.In addition to the bla KPC-2 plasmid and virulence plasmid, we also observed four copies of bla NDM-5 genes, located in two plasmids and chromosomes.Notably, though plasmid p3122-10 (bla NDM-5 ) harbored conjugation systems, it could not be self-transferred effectively, since it lost the key protein to form the membrane-associated mating pair formation complex (20).Although both two bla NDM-5 plasmids in FK3122 lose effective transmissibility, the duplication transposon mobile of bla NDM-5 genes should attract attention.Previous studies have demonstrated that increased copy numbers of bla NDM-5 genes through translocation events would contribute to cefiderocol resistance (23).In our study, we also observed the increased cefiderocol resistance level of FK3122 (four bla NDM-5 copies) compared to the FK3127 (single bla NDM-5 copy).Moreover, the co-occurrence of bla NDM-5 , bla KPC-2 , and virulence plasmids was also identified in another ST11-KL64 strain KPWX136 in east China, but which did not exhibit the high pathogenicity as the isolate obtained in this study (5).ST15 K. pneumoniae was identified as the second most frequent CRKP clone in hospital infections after ST11 K. pneumoniae, while the ST15 hypervirulent CRKP was uncommon (1).At present, there were only a few hypervirulent, and carbapenemresistant ST15 Klebsiella pneumoniae have been described, most were associated with bla OXA-232 and bla KPC-2 (24,25).In FK3127, we observed three key plasmids, p3127-6 (virulence), p3217-7(bla KPC-2 ), and p3127-8(bla NDM-5 ).Notably, all these plasmids not only could self-transfer to other isolates but also could co-transfer.Previous reports of cases of NDM-KPC-2-hv-CRKP infection are scarce, and no study found that the resistance determinants (bla NDM and bla KPC ) and virulence determinants all could be transferred in one isolate or even co-transferred.p3127-6 virulence plasmid was a typical hybrid conjugative plasmid.Previous genomic research indicated that these hybrid plasmids were most likely formed by the recombination of IncF and the virulence IncHIB Co-transfer of plasmids Co-transfer of p3127-6 and p3127-8 bla NDM-5 + iucABCD/iutA 3 2.25 × 10 −8 1.13 × 10 −8 to 1.9 × 10 −8 plasmids (26); this hybrid pattern also was confirmed in the p3127-6 plasmid.p3127-7 and p3127-8 were all typical IncFII plasmids with completed conjugation modules and effective conjugation frequencies.The co-existence of virulence plasmid and resistance plasmids facilitated the formation of hv-CPKP.Moreover, the co-existence of transmissi ble resistance and virulence plasmids in one isolate could act as a vast storage pool of resistant and virulence elements.All in all, the ST15 FK3127 posed a major threat to modern medicine, jeopardizing our ability to treat life-threatening infections.
In this study, two high-risk novel K. pneumoniae co-harboring bla NDM-5 , bla KPC-2 , and virulence plasmids were characterized.Phenotype testing and genome background analysis confirmed the increased virulence and multidrug resistance of these strains.The superbug, which combines hypervirulence and carbapenem resistance, will endanger clinical treatment.To prevent widespread transmission, tailored surveillance, antimicro bial stewardship, and strict clinical management are urgently required.

FIG 1
FIG 1 The virulence phenotypes and levels of FK3122 and FK3127.(A) Siderophores production determined by CAS agar plate.(B) The LDH activity in A549 human lung epithelial cells infected by different strains.(C) The survival curves of G. mellonella infected by FK3122, FK3127, and other control strains.(D) The survival curves of G. mellonella infected by different conjugants (E.coli).Note: 1627 (ST11 cKP, negative control), FK3036 (ST15 cKP, negative control), NS (normal saline).Unpaired two-sided Student's t-test was performed for LDH production.A log-rank (Mantel-Cox) test was performed for the survival curves.****P < 0.0001.

TABLE 1
Antimicrobial drug susceptibility

TABLE 2
General features and antimicrobial resistance genes of plasmids in K. pneumoniae FK3122 and FK3127

TABLE 3
Conjugation frequency of resistant plasmids identified in K. pneumoniae FK3127